A biological array can contain a chosen collection of biomolecules, for example, probes specific for important pathogens, sequence markers, antibodies, immunoglobulins, receptor proteins, peptides, cells, and the like. For example, an array can contain a chosen collection of oligonucleotides specific for known sequence markers of genetic diseases or probes to isolate a desired protein from a biological sample. A biological array may comprise a number of individual biomolecules tethered to the surface of a substrate in a regular pattern, each one in a different area, so that the location of the biomolecule is known.
Biological arrays can be synthesized directly on a substrate employing methods of: solid-phase chemical synthesis in combination with site-directing mass, as disclosed in U.S. Pat. No. 5,510,270, incorporated herein by reference in its entirety; photolithographic techniques involving precise drop deposition using piezoelectric pumps, as disclosed in U.S. Pat. No. 5,474,796, incorporated herein by reference in its entirety; or contacting a substrate with typographic pins holding droplets and using ink jet printing mechanisms to lay down an array matrix.
There are those who believe that generating a probe on a surface using methods of solid phase synthesis is a better process than attaching the final product to a modified surface. While this might avoid the complications of adding an anchor point (functionality added for the specific purpose of surface reaction), it produces the unavoidable consequence of any linear non-convergent synthesis which results in a low yield of the desired product. For example, a 10 step linear synthesis giving a 95% yield in each step gives a final yield of only 60%. The synthesis of a 20-mer gives a final yield of 36% and a 30-mer gives final yield of 21%. By the time a 50-mer is reached only 8% of the desired product is left. The other 92% are fragments left over during each synthesis step. An added complication is that each fragment may react in any subsequent synthetic step, which in turn generates any number of alternate sequences other than the desired one. This has the ultimate problem of producing false positives during the hybridization reaction. It would be ideal if each synthesis reaction could produce the 95% yield, which presently is not realistic because each step suffers some loss attributed to several factors which include, but are not limited to, bad reagents, wrong time and/or temperature, and contamination.
An example of a modified surface is an aldehyde surface that attaches to a primary amine to form the imine (Schiff Base), however, it requires the use of a hydride reducing agent to stabilize the bond. The reason the bond is unstable is that imines are susceptible to hydrolysis resulting in the amine and the aldehyde. Traditionally, the hydride reducing agent is a borohydride in a less reactive form like the cyanoborohydride. The purpose of the cyano group is to reduce the reactivity of the hydrides protons which immediately form H2 in the presence of water and consume the reagent. A problem with using boron is that it forms stable complexes with amine functions that usually need rigorous conditions to break. Another problem with these reducing agents is that they react with many carbonyl groups of which an aldehyde is merely one example. Amides are another type of carbonyl group present in the bases thymine, cytosine, and guanine, which can also be attacked by hydride reagents.
Substrates are available for immobilization of biomolecules using either covalent attachment of biomolecules or non-covalent attachment of biomolecules. γ-aminopropylsilane (GAPS) is traditionally used as the surface of choice for the non-covalent binding of DNA (or other biomolecules) and has given the approach a degree of success. However, better retention and signal intensity would be desirable. In particular, there is some degradation observed with the primary amine surface (decreased signal in the gold colloid test). The nature of the primary amine degradation is not well understood but is believed to be a result of a reaction with CO2 forming the carbamic acid salt or air oxidation. Further, there is an issue of whether the ammonium ions formed when using GAPS can then be covalently bonded to DNA during a UV exposure.
The present invention is directed to overcoming the above-noted deficiencies in the prior art.